Anti mcp 1 neutralizing antibody

Cell migration assays were performed using 24-well Transwell plates (Corning Costar, Pittston, PA). First, HDVSMCs were pretreated with T. pallidum at different MOIs (T. pallidum: cell ratios of 1:1, 10:1, 100:1 and 200:1) at 37°C in an atmosphere containing 5% CO₂ for 24 h in 24-well culture plates. Then, 600 μL of cell culture supernatant was collected and added to the lower chambers of Transwell plates. Approximately 2.5 ×10⁵ THP-1 cells in a volume of 200 μL were seeded in the upper chambers of the Transwell plates. The Transwell plates were then incubated at 37°C in an atmosphere containing 5% CO₂ for 2 h. THP-1 cells that migrated to the lower chamber were counted under a microscope, and the migration rate of THP-1 cells was calculated as follows: migration rate of THP-1 cells (%) = (number of THP-1 cells in the lower chamber/number of THP-1 cells added to the upper chamber) ×100. For the inhibition assays, HDVSMCs were pre-incubated with an anti-MCP-1 neutralizing antibody (30 μg/mL) (R&D Systems, Inc., Minneapolis, MN, USA) or BAY11-7082 (2 μmol/L) for 1 h. Subsequently, HDVSMCs were incubated with T. pallidum at an MOI of 100:1 for 24 h. HDVSMCs treated with phosphate-buffered saline (PBS) were used as the control cells. The migration assay was performed, and the migration of THP-1 cells was calculated as described above.